This invention relates to a process of enzymatically preparing metal proteinates and the products obtained therefrom. More specifically, this invention relates to enzymatically prepared metal proteinates wherein the proteinate is a chelate of an enzymatically digested protein with bivalent essential metal wherein the naturally occurring vitamins and hormones in the protein are not destroyed.
Heretofore the hydrolysis of proteinaceous material to form metal chelates or proteinates has utilized sufficiently strong acidic and basic reaction conditions which require the use of special equipment. Such hydrolysis results in the destruction of some of the essential amino acids forming the proteinaceous material. For example, U.S. Pat. No. 3,396,104 teaches a process whereby proteinaceous materials are subjected to a basic hydrolysis at a pH between about 8 and 10.5 and thereafter subjected to an acid hydrolysis with phosphoric acid or other strong acid in the presence of a metal salt to form a chelated amino acid complex. Similarly, U.S. Pat. No. 3,775,132 teaches a method for the production of metal proteinates wherein the proteinaceous material is first hydrolyzed by a base-acid-base hydrolysis prior to mixing with the metal salt to form the metal proteinate precipitate. These patents are disadvantageous in that hydrolysis with either acids or bases requires relatively high temperatures. Since proteins are made up from a combination of amino acids, some of the naturally occurring amino acids in the proteinaceous substances are destroyed. For example, tryptophan is destroyed in acid hydrolysis. Serine, threonine, tyrosine and several others are partially destroyed in basic hydrolysis. Not only are certain amino acids destroyed in the acid or base hydrolysis process, but also naturally occurring vitamins and hormones, which may be beneficial when ingested into the animal body, are also destroyed.
It is therefore an object of the present invention to prepare metal proteinates by the enzymatic digestion of proteinaceous material whereby naturally occurring amino acids are not destroyed.
It is a further object of the present invention to prepare metal proteinates under mild reaction conditions whereby the naturally occurring vitamins and hormones in the proteinaceous material are not destroyed.
A still further object of the present invention is to prepare metal proteinates prepared from enzymatically digested proteinaceous material whereby the hydrolysate consists of polypeptides, but wherein the hydrolysis is not carried out to the point that single amino acids are formed.
These and other objects may be accomplished by means of a novel digestion and chelation process wherein vitamins and hormones are not destroyed and no amino acids are formed under the mild conditions utilized. This leaves the proteinaceous substance in a naturally digested form which is the form that an animal body prefers. Natural digestion in the body is done by two enzymes, pepsin and pancreatin, which leaves the protein in the polypeptide form.
A particular advantage of the present invention is that the enzymatic digestion allows the formation of a substrate of mineral and protein, i.e., a metal proteinate which is essentially salt free. Most hydrolysis processes use an acid followed with a base for neutralizing. Upon spray drying these form a product, which is between 50% and 70% salt. In the present process, the filtration of the enzymatically digested metal proteinate from the water gives a metal proteinate which is essentially salt free.
Another advantage of the present invention is that in enzymatic hydrolysis, polypeptides are produced as a substrate for the chelation process. No acids or bases are used for hydrolysis, therefore no amino acids forming the polypeptides are destroyed and the hydrolysis is sufficiently mild that no single amino acid is formed.
As utilized in the present application, the term metal proteinate has reference to a metal chelate wherein a protein hydrolysate is used as the ligand thereby forming a polycyclic complex with a biologically essential bivalent metal. The coordination complex is formed between the bivalent metal and at least two polypeptide ligands, depending upon the mineral involved. For example, zinc forms Sp.sub.3 orbital hybrids in aqueous solution. This means that two polypeptide residues will chelate with zinc to form the metal proteinate. The planes of the polypeptide residue are at right angles to each other forming a tetrahedral complex. They are attached in two places to the zinc atom forming a polycylic chelated complex. Iron, on the other hand, forms octahedral complexes with three polypeptide residues. Each mineral therefore has its characteristic configuration for forming complexes depending on the oxidation state. They may have different ligands and form different configurations. Biologically essential metals which may be utilized include iron, copper, zinc, manganese, cobalt, chromium, calcium, magnesium and vanadium.
In order for chelation to take place it is essential that both the enzymatically produced protein hydrolysate and the complexing metal be in solution. The metals are therefore added in the form of soluble inorganic salts.
The protein hydrolysate is formed under mild conditions which do not result in the complete hydrolysis of the protein to the individual amino acid state, and which also does not destroy other beneficial substances in the protein such as vitamins and hormones.
The enzymatic hydrolysis is brought about by placing a comminuted protein source in an aqueous solution. In general, the solution will contain about 20% by weight/volume of proteinaceous material to water. Preferred sources of protein include naturally occurring tissues such as muscle, heart, liver, brain, pancreas, spleen, kidneys, duodenum, thymus, and orchic. As stated, these tissues can be used as carrier materials for minerals without destroying the valuable vitamins and hormones which may be destroyed by treatment with acids, bases or heat. Other protein sources such as casein, gelatin, collagen or albumin may be used.
Any protease may be utilized as the enzyme. Typical of such proteases are pepsin, pancreatin, trypsin, papain, bacterial protease and fungal protease. The enzyme is added in an amount of about 1 to 10 percent by weight based on the protein.
The hydrolysis is carried out at between about 25.degree. and 70.degree. C. so that none of the amino acids in the polypeptides are destroyed in the hydrolysis process. The hydrolysis period may last anywhere from a matter of hours to a matter of weeks depending upon the amount of hydrolysate to be formed and the ease of hydrolysis. In general, the hydrolysis will be carried out over a period of about 2 hours to about 5 days. Preferably, the hydrolysis is carried out under neutral conditions and it may be necessary to adjust the pH of the protein-enzyme digestion solution by the use of an acid or base. Small amounts of acids or bases such as hydrochloric acid and sodium hydroxide may be utilized to neutralize the enzymatic hydrolysis mixture, but not in amounts sufficient to cause acid or base hydrolysis. If desired, a small amount of preservative such as toluene may also be present during the digestion or hydrolysis process.
As the proteinaceous material is hydrolyzed into polypeptides it will be brought into solution. Preferably the hydrolysis is brought about by constantly stirring the mixture and maintaining the temperature relatively constant.
At the end of the digestion period the mixture may, if desired, be brought to a boil for a short period of time to kill the enzyme so that further hydrolysis will not take place. However, this tends to destroy natural materials such as vitamins and hormones. The solubilized hydrolysate may then be directly treated with a soluble metal salt to form a metal proteinate or may first be filtered to separate the hydrolysate from undigested tissue.
Since protein hydrolysates are chains of amino acids, it is evident that these substances will contain charged groups. The extent of such charged groups will vary with the pH and the titration curve of a protein hydrolysate will represent the composite of the effects of the various groups as they are able to combine with acids and bases. For example, in polypeptides, the charged groups may be due to amino groups belonging to the N-terminal amino acid of each polypeptide chain, or to carboxyl groups belonging to the C-terminal amino acid of a polypeptide chain. A consequence of charged groups in protein hydrolysates is the ability of the hydrolysate to form complexes with other compounds. In order to form a true metal proteinate (chelate) one must have the proper amount of constituents at the right conditions. It is essential that the mineral and protein hydrolysate both be in soluble form. It is also important that the protons be removed from the carboxyl groups before a chemical bond with the mineral can be formed. In the case of protein hydrolysates, both the amino and carboxyl functions must be free from interfering protons in order for chelation to take place. With protein hydrolysates, such as polypeptides, most protons remain intact in neutral solutions and therefore the pH is preferably adjusted in a range more basic than a pH of 7.5. Preferably the pH will be adjusted to a point that is on the alkaline side of the isoelectric point. In general, pHs of about 7.5 to 10 are preferred. Since the hydrolysate has previously been formed, the pH adjustment does not materially affect the stability of the hormones and vitamins in the protein hydrolysate. The appropriate amount of metal salt is added to an aqueous solution of the protein hydrolysate to form a precipitate which is filtered and then washed. The amount of metal salt that is added is adjusted such that there are at least 2 moles of protein hydrolysate or polypeptide per mole of metal salt. Otherwise, a true chelate will not be formed. The metal chelates readily precipitate, and unlike the protein hydrolysates, do not have a net charge. In other words, there are no free ions associated with the metal proteinates.
The product thus obtained can be dried and mixed in appropriate amounts as a dietary supplement in the form of a powder, tablet, liquid or in any other form desired, and then administered to an animal having a need for the particular metal proteinate thus formed, or if desired the metal proteinates may be mixed in an appropriate base for topical or cosmetic application.
It becomes at once apparent that many different metal proteinates can be formed according to the present invention and that the particular metals used can be used in predetermined amounts. The Amount and source of proteinaceous material enzymatically digested can be predetermined so that the vitamins and hormones which will be present in the final product can also be predicted. A distinct advantage of the present invention is that the metals, vitamins and hormones are delivered to the body utilizing a proteinaceous substrate in a naturally digested form which is the form most readily accepted and assimilated by the body.